Pegylated obese (ob) protein compositions

ABSTRACT

Polyethylene and polypropylene protein conjugates which modulate body weight of animals and humans for the treatment, prevention and control of obesity and associated diseases or conditions, and the recombinant expression of these biologically active proteins in purified and homogeneous forms.

This application is a continuation-in-part of application Ser. No.08/484,629, filed Jun. 7, 1995 which is a continuation-in-part ofapplication Ser. No. 08/435,777 filed May 5, 1995.

BACKGROUND

Obesity is reported to be the commonest nutritional disorder in Westernsocieties (Zhang, Y. et al. (1994)) More than three in 10 adultAmericans weigh at least 20% in excess of their ideal weight (Zhang, Y.et al (1994)). Increased body weight is a public health problem becauseit is associated with important medical morbidities such as type IIdiabetes mellitus (i.e., non-insulin-dependent diabetes mellitus),hypertension and hyperlipidaemia (Crundy, S. M. & Barnett (1990)). Thereis evidence that body weight is physiologically regulated and theobesity (and its related conditions or diseases) are due in part toderangements in this regulation (Zhang, Y. et al. (1994)).

In rodents, there are described seven single gene mutations that resultin an obese phenotype; five of which are present in mice. Of these sevenrodent models, one of the most intensively studied is the obese (ob)gene mutation in mice, identified in 1950 (Ingalls, A. M. et al.(1950)). Mice homozygous for this ob gene mutation are profoundly obese,develop type II diabetes mellitus, and are hyperphagic andhypometabolic, as part of a syndrome resembling morbid obesity in man(Friedman J. M. et al. (1991)). This ob gene is mapped to the mouseproximal chromosome 6 and encodes a protein (i.e., ob protein) expressedin adipose tissue (Zhang, et al. (1994)). Mice homozygous for the obgene mutation have little to no production of this ob protein, andaccordingly have defective regulation of body weight leading to obesity.

The murine or human ob proteins may be administered to patientssuffering from defects or mutations in their corresponding obese (ob)gene, which defects or mutations prevent or interfere with theproduction and/or function of the ob proteins in modulating body weight.These proteins may therefore be used as a hormone-like substance tocontrol, prevent or treat obesity and its related diseases andconditions in man and animals.

To use the murine or human ob proteins in this manner, these proteinscan be administered through injection by a variety of routes, such asintraperitoneal, intravenously, intramuscularly or subcutaneously, infrequent dosages. Since it is administered frequently through injection,it is important that the murine or human ob proteins be purified,preferably to homogeneity, be free of contaminating protein materials,and be recombinantly expressed in a soluble and biologically activeform. It is generally known to practitioners in the field thatcontaminants present in injectable medication can often lead to toxicside-effects or adverse immunological responses.

While the murine ob gene sequence is disclosed in Zhang, et al (1994),no methods of expressing the murine ob protein or its human counterparthave been reported, much less producing these proteins in a biologicallyactive and soluble state from which the proteins can be purified tohomogeneity. Therefore it is important, and is an object of thisinvention, to express and produce the murine or human ob proteins in ahomogeneous, soluble, and biologically-active state.

SUMMARY OF THE INVENTION

It has been discovered that recombinant human and murine ob proteins canbe expressed in a biologically active and soluble state, and thereafterpurified to homogeneity suitable for injection to patients for treating,preventing or controlling obesity and its related conditions anddiseases, such as type II diabetes mellitus, hypertension,hyperlipidaemia and the like.

In accordance with this invention, the human and murine ob proteins canbe produced recombinantly in a biologically active form and purified tohomogeneity by first constructing novel expression vectors forEscherichia coli (E. coli). These expression vectors contain a promoterand a DNA sequence, which DNA sequence encodes a fusion proteincomprising two parts: the signal peptide of the outer membrane protein Aof E. coli (i.e., sOmpA) and the human or murine ob protein. Inaccordance with this invention, the next step for producing thebiologically active recombinant form of the murine and human ob proteinsis to insert this expression vector in an E. coli host whereby there isobtained efficient expression and translocation of the fusion proteininto the periplasmic space (i.e., between the inner and outer cellmembranes of the E. coli microorganism), at which point the signalpeptide is excised from the ob protein leaving the ob protein in asoluble and biologically active form.

The present invention is further directed to 1) an expression vectorcontaining the DNA encoding a fusion protein comprising a sOmpA signalpeptide and a human or murine ob protein; 2) to a host organismtransfected or transformed by such expression vector; and 3) to the DNAsequence encoding the human ob protein and 4) polyethylene orpolypropylene glycol conjugates of the ob protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the two clones for human ob protein; i.e., hob1and hob2, which schematics depict the location and types of restrictionsites located at the 5' and 3' ends of the human ob cDNA sequence.

FIG. 2 is a schematic of the construction of the pLPPsOmpA mobexpression vector.

FIG. 3 is a schematic of the construction of the pLPPsOmpA hob1expression vector.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered a method for expressing recombinant human andmurine ob proteins in a biologically active and soluble state, and forproducing these proteins in a purified homogeneous form suitable foradministration to animals and humans. The recombinant human and murineob proteins of this invention can be conjugated with polyethylene orpolypropylene glycol homopolymers.

The human and murine ob proteins can be produced recombinantly in abiologically active state in accordance with this invention by firstconstructing novel expression vectors for E. coli containing a promoterand a DNA sequence, which DNA sequence encodes a fusion proteincomprising the signal peptide of the outer membrane protein A of E. coli(i.e., sompA) and the human or murine ob protein. The next step forobtaining the murine or human ob protein in a biologically active andsoluble form is to insert this expression vector in an E. coli hostwhereby there is obtained efficient expression and translocation of thefusion protein into the periplasmic space (i.e., between the inner andouter cell membranes of the E. coli). Once in the periplasmic space, thesignal peptide is excised from the ob protein leaving the ob protein ina soluble and biologically active form. Next, the ob proteins areefficiently secreted in soluble and biologically active form into cellfree medium following treatment of the host E. coli cells to coldosmotic shock, at which point the ob proteins are purified tohomogeneity by the sequential use of anion exchange chromatography,hydrophobic interaction column chromatography and gel filtration,carried out in that order.

Murine Ob Gene and Murine Ob Protein

The method for expressing and producing the murine ob protein inaccordance with this invention is achieved utilizing the murine ob geneas reported by Zhang et al (1994), the sequence for which gene is a 702base pair (bp) nucleotide sequence identified herein as SEQ ID NO. 1.This murine ob gene sequence comprises a 501 bp coding sequence or openreading frame (ORF) starting with a start codon at nucleotide 36 andterminating with a stop codon at nucleotide 537, and having untranslatedsequences at both the 31 and 5' ends. The ORF contains a 63 bp signalsequence from nucleotide 36 to 98.

This murine ob gene sequence (SEQ ID NO: 1) encodes the murine obprotein (plus its signal sequence) whose amino acid sequence is 167amino acids in length and is identified as SEQ ID NO: 2. In this proteinof SEQ ID NO: 2, the first 21 amino acids represent the signal sequenceof the murine ob protein. The mature murine ob protein (without itssignal sequence) extends from amino acid 22 (Val) to amino acid 167(Cys) and is represented by SEQ ID NO: 3.

The Human Ob Gene and Human Ob Protein

The method for expressing and producing the human ob protein inaccordance with this invention is achieved utilizing the human ob gene,the sequence for which gene is a 690 bp nucleotide sequence identifiedherein as SEQ ID NO: 4.

Zhang et al (1994) reports the human ob gene as highly homologous to themurine ob gene, and discloses a conventional method usingoligonucleotide probes directed to the murine ob gene which can beutilized to 1) screen a cDNA library of clones derived from humanadipose tissue, 2) identify those clones having the human ob gene, and3) isolate and sequence the human ob gene sequence. When sequenced byconventional means, this human ob gene sequence is determined to havethe nucleotide sequence SEQ ID NO: 4.

As with the murine ob gene, the human ob gene comprises a 501 bp codingsequence or open reading frame (ORF) starting with a start codon atnucleotide 37 and terminating with a stop codon at nucleotide 538, andhaving an untranslated sequences at both the 3' and 5' ends. The ORFcontains a 63 bp signal sequence from nucleotide 37 to 99.

This human ob gene sequence (SEQ ID NO: 4) encodes a human ob proteinplus its signal sequence whose amino acid sequence of 167 amino acids inlength is identified as SEQ ID NO: 5. The first 21 amino acids of thisprotein of 167 amino acids in length represent the signal sequence. Themature human ob protein (without its signal sequence) extends from aminoacid 22 (Val) to amino acid 167 (Cys) and is represented by SEQ ID NO:6.

Zhang et al (1994) reports 84% identity between the murine and human obproteins. Zhang et al (1994) also reports that variants of the murineand human proteins exist, one such variant being characterized in bothspecies by a deletion of glutamine 49. Approximately 30% of cDNA clonesin the libraries derived from mouse adipose tissue and human adiposetissue have the codon 49 missing (Zhang et al (1994)).

Definitions

The following terms shall have the definitions set out below:

Murine ob protein (mob) refers to the protein of SEQ ID NO: 3 whosebiological properties relate to the treating, controlling or preventingobesity or its associated conditions and diseases. Specifically, amurine ob protein is defined to include any protein or polypeptidehaving an amino acid sequence which is substantially homologous to theamino acid sequence SEQ ID NO: 3, and further having the followingbiological activities:

1) When the protein or polypeptide is administered byintracerebroventricular (ICV) injection to 16-18 hour fasted matureobese ob/ob mice having a body weight of at least 30 grams at a dose of20 ug or less using the methods of Haley and McCormick (1957), theprotein or polypeptide:

(a) reduces food intake during a 5 hour feeding test by 50% compared tovehicle injected control mice (ED50 for reducing food intake); and

(b) reduces body weight gain during the 24 hours following the ICVinjection by at least 50% compared to vehicle injected control mice(ED50 for reducing body weight gain);

or

2) When the protein or polypeptide is administered intraperitoneal (IP)to non-fasted mature ob/ob mice having a body weight of at least 30grams twice a day at the beginning of daylight and again at the 3 hourpoint of the dark phase, for one week, in a total daily dose of 20 ug orless, the protein or polypeptide:

(a) reduces 5 and 24 hour food intake by at least 20% compared tovehicle injected control mice (ED20 for reducing food intake); and

(b) reduces body weight gain during the 24 hours following the first IPinjection by at least 20% compared to vehicle injected control mice(ED20 for reducing body weight gain).

As used herein the term murine ob protein includes such proteinsmodified deliberately, as for example, by site directed mutagenesis oraccidentally through mutations.

Human ob protein (hob) refers to the protein of SEQ ID NO: 6 whosebiological properties relate to the treating, controlling or preventingobesity or its associated conditions and diseases. Specifically, a humanob protein is defined to include any protein or polypeptide having anamino acid sequence which is substantially homologous to the amino acidsequence SEQ ID NO: 6, and further having the following biologicalactivities:

1) When the protein or polypeptide is administered ICV to 16-18 hourfasted mature obese ob/ob mice having a body weight of at least 30 gramsat a dose of 20 ug or less using the methods of Haley and McCormick(1957), the protein or polypeptide:

(a) reduces food intake during a 5 hour feeding test by 50% compared tovehicle injected control mice (ED50 for reducing food intake); and

(b) reduces body weight gain during the 24 hours following the ICVinjection by at least 50% compared to vehicle injected control mice(ED50 for reducing body weight gain);

or

2) When the protein or polypeptide is administered IP to non-fastedmature ob/ob mice having a body weight of at least 30 grams twice a dayat the beginning of daylight and again at the 3 hour point of the darkphase, for one week, in a total daily dose of 20 ug or less, the proteinor polypeptide:

(a) reduces 5 and 24 hour food intake by at least 20% compared tovehicle injected control mice (ED20 for reducing food intake); and

(b) reduces body weight gain during the 24 hours following the first IPinjection by at least 20% compared to vehicle injected control mice(ED20 for reducing body weight gain).

As used herein the term human ob protein includes such proteins modifieddeliberately, as for example, by site directed mutagenesis oraccidentally through mutations.

Substantially homologous which can refer both to nucleic acid and aminoacid sequences, means that a particular subject sequence, for example, amutant sequence, varies from a reference sequence by one or moresubstitutions, deletions, or additions, the net effect of which do notresult in an adverse functional dissimilarity between the reference andsubject sequences. For purposes of the present invention, sequenceshaving greater than 95 percent homology, equivalent biologicalproperties, and equivalent expression characteristics are consideredsubstantially homologous. For purposes of determining homology,truncation of the mature sequence should be disregarded. Sequenceshaving lesser degrees of homology, comparable bioactivity, andequivalent expression characteristics are considered substantialquivalents. Generally, homologous DNA sequences can be identified bycross-hybridization under standard hybridization conditions of moderatestringency.

Fragment of the murine or human ob protein means any protein orpolypeptide having the amino acid sequence of a portion or fragment of amurine or human ob protein, and which has the biological activity of themurine or human ob protein, respectively. Fragments include proteins orpolypeptides produced by proteolytic degradation of the murine or humanob proteins or produced by chemical synthesis by methods routine in theart.

An ob protein or fragment thereof is biologically active whenadministration of the protein or fragment to a mammal, including man,reduces food intake and reduces the rate of weight gain in the mammal.Determining such biological activity of the human or murine ob proteincan caried out by conventional, well known tests utilized for suchpurposes on one or more species of mammals, particularly the obese ob/obmouse. Several of these tests which can be utilized to demonstrate suchbiological activity are described herein. In determining biologicalactivity in accordance with the ICV test in ob/ob mice as describedherein, the human or murine ob protein preferably has an ED50 forreducing food intake of 20 ug or less and an ED50 for reducing bodyweight gain of 20 ug or less. Alternatively, in determining biologicalactivity of the human or murine ob protein in accordance with the IPtest in ob/ob mice as described herein, the human or murine ob proteinpreferably has an ED20 for reducing food intake of 20 ug or less and anED20 for reducing body weight gain of 20 g or less. Generally, fragmentswhich exhibit the above mentioned biological activity are preferred.

Replicon is any genetic element (e.g., plasmid, chromosome, virus) thatfunctions as an autonomous unit of DNA replication in vivo, i.e.,capable of replication under its own control.

Expression vector is a replicon, such as a plasmid, phage or cosmid, towhich another DNA segment may be attached so as to bring about thereplication of the attached segment. It comprises a transcriptional unitcomprising an assembly of (1) a genetic element or elements having aregulatory role in gene expression, for example, promoters or enhancers,(2) a structural or coding sequence which is transcribed into mRNA andtranslated into protein, and (3) appropriate transcription initiationand termination sequences.

Clone is a group of DNA molecules derived from one original length ofDNA sequences and produced by a bacterium or virus using geneticengineering techniques, often involving plasmids.

Signal sequence is the nucleic acid sequence located at the beginning(5' end) of the coding sequence of a protein to be expressed. Thissignal sequence encodes a signal peptide, N-terminal to the newlysynthesized protein, that directs the host cell to translocate theprotein toward or through the host cell membrane, and which signalpeptide is usually excised during such translocation.

Start codon is a codon usually ATG located in the coding sequence of aprotein, and usually at the 5' end, and signals the first amino acid ina protein sequence.

Stop codon is a nonsense codon located in and usually at the 3' end of acoding sequence of a protein, and signals the end of a growingpolypeptide chain.

Open Reading Frame (ORF) is a linear array of codon triplets indouble-stranded DNA encoding an amino acid sequence in a cell in vitroor in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the ORF are determined by a start codon atthe 5' terminus and a stop codon at the 3' terminus. It may be referredto as a "coding sequence".

Promoter sequence is DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3'direction) open reading frame of one or more structural genes. Thepromoter sequence is usually located at the 5' end of the signalsequence or open reading frame and extends upstream in the 5' directionto include the minimum number of bases or elements necessary to initiatetranscription of the polypeptide at a level detectable above background.

A coding sequence or ORF is under the control of a promoter sequencewhen RNA polymerase transcribes the coding sequence into mRNA.

A composition comprising A (where A is a single polypeptide) ishomogeneous for A when there is no detectable quantity of contaminatingproteins or other endogenous materials, as detected by conventionalmeans, for example, staining of polyacrylamide gels. For purposes ofthis invention, the term homogeneous shall refer to a compositioncomprising a single protein or polypeptide when at least 95% by weightof the composition is that single protein or polypeptide.

Method for Expressing Ob Proteins

The following steps outline the methods for recombinantly expressing thehuman and murine ob proteins in a biologically active and solublecell-free state, free of other mammalian proteins, from which the obproteins can then be purified to homogeneity. These steps areexemplified in detail in the examples.

1) Obtaining the Mouse and Human Ob Genes

The cDNA (SEQ ID NO. 1) encoding the murine ob protein plus its naturalsignal sequence is published in Zhang et al. (1994). This murine cDNAhas been isolated and amplified by PCR technique usingoligodeoxynucleotide DNA primers by conventional techniques. These DNAprimers and the methods for obtaining them are described in Zhang et al(1994).

The cDNA (SEQ ID NO. 4) encoding the human ob protein plus its naturalsignal sequence is obtained using the same oligodeoxynucleotide DNAprimers as used in Zhang et al (1994) to obtain the murine ob gene. Byusing conventional technique, this human cDNA has been isolated from alambda phage cDNA library made from RNA derived from human adipocytetissue.

The human or mouse ob cDNA may be obtained not only from cDNA libraries,but by other conventional means; e.g., by chemical synthesis, or bycloning genomic DNA, or fragments thereof, purified from the desiredcell. These procedures are described in Sambrook, et al. (1989), DNACloning: A Practical Approach (1985), Benton and Davis (1977), andGrunstein and Hogness (1975). To obtain the human or mouse ob cDNA fromcDNA libraries, the cDNA libraries are screened by conventional DNAhybridization techniques by the methods of Benton and Davis (1977) orGrunstein and Hogness (1975) using primers prepared by reversetranscription of polyadenylated RNA isolated from murine adipose cellscontaining the murine ob gene. Clones which hybridize to the primers areanalyzed by restriction endonuclease cleavage, agarose gelelectrophoresis, and additional hybridization experiments ("Southernblots") involving the electrophoresed primers. After isolating severalclones which hybridized to the murine cDNA probes, the hybridizingsegment of one clone is subcloned and sequenced by conventionaltechniques.

Clones derived from genomic DNA may contain regulatory and intron DNAregions in addition to coding regions: clones derived from cDNA will notcontain intron sequences. In the molecular cloning of the gene fromgenomic DNA, DNA fragments are generated, some of which will encode thedesired gene. The DNA may be cleaved at specific sites using variousrestriction enzymes. Alternatively, one may use DNAse in the presence ofmanganese to fragment the DNA, or the DNA can be physically sheared, asfor example, by sonication. The linear DNA fragments can then beseparated according to size by standard techniques, including but notlimited to, agarose and polyacrylamide gel electrophoresis and columnchromatography.

Whatever the source, the human or murine ob gene may be molecularycloned into a suitable vector for propagation of the gene by methodsknown in the art. Any commercially available vector may be used. Forexample, the mouse cDNA may be inserted into a pCDNA3 vector and thehuman cDNA may be inserted into a pBluescriptSK⁻ vector. Appropriatevectors for use with bacterial hosts are described by Pouwels, et al(1985). As a representative but nonlimiting example, useful cloningvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmids whichare in turn derived from the well known cloning vector pBR322 (ATCC37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec,Madison, Wisc., USA).

The nucleotide sequences of the human or murine ob gene inserted inthese commercially available vectors can be verified by methods known inthe art, by standard nucleotide sequencing techniques.

Other nucleic acids that code for ob proteins of species other thanhuman or murine may be used herein. Accordingly, while specific DNA hasbeen cloned and sequenced in relation to the human and mouse ob gene,any animal adipocyte potentially can be used as the nucleic acid sourceof the ob protein.

2) Construction of an Expression Vector for the Human and Murine ObProtein

The human or murine ob gene cloned in accordance with the methodsdescribed above are used to construct the expression vectors for thehuman and murine ob proteins, respectively.

For expression of the biologically active human and murine ob protein bya transfected or transformed E. coli host cell and for secretion of theob protein into the periplasm, a novel expression vector can beutilized. This expression vector includes a promoter and a DNA sequenceencoding a fusion protein. The fusion protein consists of two parts: thefirst part being a signal peptide for the outer membrane protein A of E.coli (sOmpA) and the second part of the fusion protein being the humanor murine ob protein (minus their own natural signal sequences). The DNAsequence encoding this fusion protein also consists of two parts: afirst part that encodes the sOmpA peptide and a second part that encodesthe murine or human ob protein (minus their natural signal sequences).The first part of the DNA sequence that encodes the sOmpA peptide is thesignal sequence described by De Sutter, K. et al (1994) and has thenucleotide sequence of SEQ ID NO: 7. The second part of the two-part DNAsequence encodes the murine or human ob proteins and has the nucleotidesequence of SEQ ID NO: 1 or SEQ ID NO: 4, respectively minus thatportion of the nucleotide sequence that encodes the respective naturalsignal sequences.

The signal peptide encoded by the sOmpA signal sequence of SEQ ID NO: 7has the amino acid sequence SEQ ID NO: 8 as reported by De Sutter, K. etal (1994).

The novel expression vector of this invention is achieved by insertingthe promoter and DNA sequence encoding the fusion protein into aconventional expression vector suitable for expression of recombinantproteins in E. coli host cells.

In constructing this novel expression vector in accordance with thisinvention, any promoter may be used as long as it is capable ofcontrolling transcription of the fusion protein comprising the sOmpApeptide and the ob protein in the E. coli host cell. When the sOmpA isused as the signal peptide, it is preferable to use both thelac-operator promoter (PO_(lac)) and the lipoprotein promoter (P_(lpp)).Other useful promoters for such expression in E. coli include the T7 RNApolymerase promoter described by Studier et al, (1986), the laczpromoter described by Lauer, (1981) and available as ATCC 37121, the tacpromoter described by Maniatis, (1982) and available as ATCC 37138, thealkaline phosphatase (phoA) promoter, and the trp promoter described byGoeddel et al. (1980). Other promoters have been discovered and utilizedin E. coli and details concerning their nucleotide sequences, enabling askilled worker to ligate them functionally within the expression vectorof this invention, have been published (Siebenlist et al.(1980).

Next, the method for constructing this novel expression vector isdescribed. This method is further detailed in the Examples and depictedin FIGS. 1 and 2. First, the coding sequence of the human or mouse obgene (minus its natural signal sequence) is incorporated into a plasmidcontaining the sOmpA signal sequence, such as the plasmid pT10sOmpArPDI.This pT10sOmpArPDI plasmid and its construction and preparation isdescribed by De Sutter K. et al (1994). Once incorporated into thisplasmid, the human or mouse ob gene is fused to this sOmpA gene tocreate a "hybrid gene sequence" in this plasmid. The sOmpA gene must beupstream of the 5' region of the ob gene coding sequence. Thereafter,promoters as enumerated above, and preferably the lipoprotein promoter(P_(lpp)) and the lac promoter-operator (PO^(lac)), are incorporatedinto this plasmid containing the hybrid gene sequence to create theexpression vectors of this invention. Two embodiments of theseexpression vectors are identified as pLPPsOmpA mob and pLPPsOmpA hob1and are depicted in FIGS. 1 and 2, respectively.

Any method or procedure known in the art to construct such a plasmid maybe used. Moreover, the order by which one fuses the sOmpA and ob genesequences, incorporates the gene sequences into a suitable plasmid, andincorporates the promoter to arrive at the expression vector of thisinvention is not critical. For example, the sOmpA gene sequence can beinitially fused to the murine or human ob gene sequence directly tocreate a hybrid gene sequence, and then this hybrid sequence insertedinto a plasmid having already incorporated therein the appropriatepromoters. It is necessary however that the sOmpA gene sequence beupstream at the 5' end of the murine or ob gene sequence.

We have discovered that by using such novel expression vector, and inparticular, by using the signal sequence encoding the sOmpA, the murineor human ob proteins can be translocated to the periplasmic space, wherethe signal peptide is appropriately cleaved leaving intact the human ormurine ob proteins therein in a soluble and biologically active form.Once in this periplasmic space, the ob proteins are efficiently secretedto the cell free environment free of other mammalian proteins uponsubjecting the host cells to cold osmotic shock, at which time the obproteins can be purified to homogeneity in a biologically active form.

3. Expressing the Human or Murine Ob Proteins in Transformed E. colicells

Next, the expression vectors constructed in accordance with the abovedescribed procedures are inserted into a host E. coli cell to transformthe E. coli cell. Any strain of E. coli may be used, such as E. coliK-12 strain 294 as described in British Patent Publication No. 2055382 A(ATCC No. 31446). Other strains useful in accordance with this inventioninclude E. coli MC1061 (Casadaban and Cohen (1980)), E. coli B, E.coli×1776 (ATTC No. 31537), and E. coli W 3110 (ATCC No. 27325) or otherstrains many of which are deposited and available from recognizedmicroorganism depository institutions.

The transformed E. coli cells are grown to an appropriate cell densityand cultured by standard methods. In so growing and culturing thetransformed E. coli hosts, the expression vectors of this inventionefficiently and effectively allow expression of the murine or human obproteins and translocation of these same proteins into the periplasm ofthe host E. coli cells in a soluble and biologically active form. ThesOmpA signal peptide (i.e., part 1 of the fusion protein) is cleavedduring translocation of the fusion protein into the periplasm yieldingthe biologically active ob protein free of other mammalian proteins orpolypeptides.

Methods for Purifying Human or Murine Ob Proteins

The recombinantly produced human or murine ob proteins in a solublebiologically active state in the periplasm of transformed E. coli cellsare thereafter purified to homogeneity.

1) Obtaining Human or Murine Ob Protein in a cell-free State Free ofother Mammalian Proteins or Polypeptides.

The recombinant human and murine ob proteins translocated to the cellperiplasm in accordance with the procedures described herein can beeffectively secreted outside the cell by subjecting the host cells tocold osmotic shock by methods known in the art and described byKoshland, D. and Botstein, D. (1980). The use of cold osmotic shockliberates from the E. coli the ob proteins in their biologically activestate free of other mammalian proteins or polypeptides.

2) Purification to Homogeneity of Human or Murine Ob Proteins

The human or murine ob proteins located in the osmotic fluid followingcold osmotic shock of transformed E. coli cells, in accordance with theabove described procedure, are biologically active and can be purifiedto homogeneity using a combination of anion exchange columnchromatography, hydrophobic interaction column chromatography and gelfiltration. Anion exchange and hydrophobic interaction chromatographycan be carried out in any order, however, the use of either must precedegel filtration.

The anion exchange stage can be carried out by conventional means. Thepreferred column for anion exchange chromatography is a Q Sepharose FastFlow column. Suitable anion exchange chromatography media includevarious insoluble matrices comprising diethylaminoethyl (DEAE) ordiethyl-(2-hydroxypropyl)aminoethyl (QAE) groups. The matrices can beacrylamide, agarose, dextran, cellulose or other types commonly employedin protein purification. A particularly useful material for anionexchange chromatography is DEAE-Sephacel Sephacel (Pharmacia). Whenmedia containing DEAE groups are employed, extracts containing murine orhuman ob proteins are applied at a weakly basic pH; e.g., pH 8.1. Thebound murine or human ob proteins can be eluted in more highly purifiedform by application of a salt gradient in a suitable buffer such asTris-HCl. Generally, the characteristics of the gradient can bedetermined by preliminary elution experiments involving a small quantityof recombinant protein.

The material containing the human or murine ob protein obtained throughthe use of anion exchange chromatography, when anion exchangechromatography is used as the first stage of purification, is nextsubjected to hydrophobic interaction chromatography. Hydrophobicinteraction chromatography is a separation technique in which substancesare separated on the basis of differing strengths of hydrophobicinteraction with an uncharged bed material containing hydrophobicgroups. Typically, the hydrophobic interaction column is firstequilibrated under conditions favorable to hydrophobic binding, e.g.,high ionic strength. A descending salt gradient may be used to elute thesample.

Any hydrophobic interaction column can be used. The preferredhydrophobic column is phenyl Sepharose; however butyl Sepharose can alsobe utilized. In accordance with the invention, the material containingthe recombinant murine or human ob protein which has been eluted fromthe anionic column is loaded onto a column containing a relativelystrong hydrophobic gel such a phenyl sepharose. To promote hydrophobicinteraction with the hydrophobic gel, a solvent is used which contains,for example, greater than or equal to 0.4 M ammonium sulfate, with 0.4 Mbeing preferred. Thus the column and the sample are adjusted to 0.4 Mammonium sulfate in 50 mM Tris buffer and the sample applied to thecolumn. The column is washed with 0.4 M ammonium sulfate buffer. The obprotein is then eluted with solvents which attenuate hydrophobicinteractions such as, for example, decreasing salt gradients, ethyleneor propylene glycol, or urea. A preferred embodiment involves washingthe column sequentially with the Tris buffer and the Tris buffercontaining 20% ethylene glycol. The ob protein is subsequently elutedfrom the column with a gradient of decreasing ammonium sulfateconcentration and increasing ethylene glycol concentration in the Trisbuffer. The collective and sequential use of anion exchangechromatography and hydrophobic interaction column chromatography, in anyorder, yields human or murine ob protein routinely at an estimatedpurity of 90%.

The gel filtration chromatography step follows the anion exchangechromatography and hydrophobic interaction column chromatography stepsoutlined above, and can be performed by any conventional gel filtrationprocedure. The ob protein eluted from the hydrophobic interactioncolumn, or the anion exchange column, whichever column is used last, canbe concentrated and dialyzed to a small volume by using a membrane witha cut-off molecular weight of 10,000 (AMICONYM10 membrane). Theconcentrated material can then be loaded onto a column containing gelfiltration media such as G100-Sephadex (Pharmacia). The ob protein canthen be separated from other contaminants on the basis of its molecularweight by standard techniques using SDS-PAGE.

The collective and sequential use of anion exchange chromatography,hydrophobic interaction column chromatography and gel filtrationroutinely yields human or murine ob protein at 95% purity.

3) Sequence Analysis N terminal amino acid sequencing of the purifiedmurine or human ob protein can be performed by methods known in the art;e.g., by electrotransfer according to the methods of Laemli, U. K.(1970) or by the procedures described by Matsudaira, P., (1987).Internal sequencing can also be done by methods known in the art. Forexample, peptide fragments may be generated by digesting the M band (onnitrocellulose) with endoproteinase Lysine C and then separated by anHPLC system.

Biological Assays for Human or Murine Ob Proteins

The biological activity of the purified human and murine ob proteins ofthis invention are such that frequent administration of the ob proteinby injection to human patients or mice result in decreased food intakeand decreased rate of weight gain compared to non-injected or controlgroups of subjects.

The biological activity of the human and murine ob proteins, orfragments thereof, obtained and purified in accordance with thisinvention can be tested by routine methods; e.g., by repeated or singleintracerebroventricular (ICV) injection in ob/ob mice according to theprocedures of Haley, T. J. et al (1957) as described in detail inExample 10. Based on this ICV test, the ED50 for reducing food intakeand the ED50 for reducing body weight gain can be determined. Inaddition, the biological activity of the purified human and murine obproteins or fragments thereof can be determined by repeated IP injectionin ob/ob mice as detailed in Example 11. Based on the IP test, the ED20for reducing food intake and the ED20 for reducing body weight gain canbe determined.

The biological activity of the human and murine ob proteins, orfragments thereof, obtained and purified in accordance with thisinvention can also be determined in humans by methods known in the art;e.g., measuring the reduction of test meal intake following IVadministration of the ob protein to the obese human test subjectscompared to IV administration of saline control, in accordance with themethods of Muurahainen, N. E., et al (1991), and as described in detailin Example 12. Alternatively, the ability of the purified murine andhuman ob proteins of this invention to reduce the rate of weight gain(e.g., induce weight loss) can be determined by repeated IVadministration to obese human test subjects according to the methods ofDrent, M. L. et al (1995), as described in detail in Example 13.

The murine and human ob proteins of this invention when purified inaccordance with this invention have biological activity in that:

1) When they are administered by intracerebroventricular (ICV) injectionto 16-18 hour fasted mature obese ob/ob mice having a body weight of atleast 30 grams at a dose of 20 ug or less using the methods of Haley andMcCormick (1957), the protein or polypeptide:

(a) reduces food intake during a 5 hour feeding test by 50% compared tovehicle injected control mice (ED50 for reducing food intake); and

(b) reduces body weight gain during the 24 hours following the ICVinjection by at least 50% compared to vehicle injected control mice(ED50 for reducing body weight gain);

and

2) When they are administered intraperitoneal (IP) to non-fasted matureob/ob mice having a body weight of at least 30 grams twice a day at thebeginning of daylight and again at the 3 hour point of the dark phase,for one week, in a total daily dose of 20 μg or less, the protein orpolypeptide:

(a) reduces 5 and 24 hour food intake by at least 20% compared tovehicle injected control mice (ED20 for reducing food intake); and

(b) reduces body weight gain during the 24 hours following the first IPinjection by at least 20% compared to vehicle injected control mice(ED20 for reducing body weight gain).

In addition this reduction in body weight and food intake even takeplaces at doses below 20 ug or less, even at a dosage level administeredICV of 1 μg or less especially when these proteins are purified tohomogenity.

The biological assays described above and detailed in the examples fordetermining the biological activity of human and/or murine ob proteinscan be used to determine the biological activity of fragments of theseproteins, whether these fragments are produced by proteolyticdegradation of the ob proteins, by chemical synthesis by recombinantprotein expression of a portion DNA sequence for the OB proteins or byany other means known to the skilled artisan.

In accordance with an embodiment of this invention, the murine and humanOB protein of this invention can be conjugated with polyethylene orpolypropylene glycol homopolymers which can be unsubstituted orsubstituted by etherification of the one of the hydroxy groups at one ofits ends with a lower alkyl group. These conjugates provide the OBprotein in stable form and improve the half life of these proteins. Inaddition, the use of these conjugates formed from polyethyleneglycol orpolypropylene glycol homopolymers provide means for increasing the halflife of the activity of the OB protein in the body. Furthermore, theseconjugates have been found to provide additional advantages such asincreasing the stability and circulation time of the therapeutic OBprotein in the body while also decreasing the immunogenicity of the OBprotein. These pegylated OB proteins can also be readily adsorbed in thehuman body and provide increased uptake in the blood system.

The preferred polyethylene or polypropylene glycol homopolymers whichare conjugated to the OB protein have molecular weights of approximately15 to 60 kDa, to produce a protein which can be mono- or poly-pegylatedwith polyethylene or polypropylene glycol molecules. In the preferredcase, the OB protein is either mono- or di-pegylated to form a conjugatewith polyethylene or polypropylene glycol units, which units in theconjugate have a total molecular weight of from 15 to 60 kDa, mostpreferably from 35 to 45 kDa. In general, the conjugates are produced asmixture of polyethylene and polypropylene glycol conjugates sincepolyethylene and polypropylene glycol starting materials are sold as amixture of different homopolymers having different molecular weights.The molecular weight set forth above is average molecular weight of themixture of OB conjugates thus produced. These mixtures can be separatedinto the individual conjugates, if desired, by conventional means suchas by column chromatography which includes HPLC. However, for treatment,generally this conjugate is utilized as a mixture The polyethyleneglycolor polypropylene glycol polymers [PEG] can be attached to the OB proteinvia the free N-terminal amino acid of the protein to form the conjugateby any conventional means. Methods for attachment of the polyethylene orpolypropylene glycol to form the conjugates with the OB protein can beby any of the many number of known methods available. The polyethyleneor polypropylene glycol may be covalently bonded through the N-terminalamino acid of the protein, as well as also through the various lysineresidues on the OB protein.

Additionally, the polyethyene or polypropylene glycol homopolymers maybe conjugated to the OB protein by bi- or poly functional linkinggroups. In producing mono-polyethylene or polyproplylene gylcolhomopolymer conjugates, di-functional linkers are used and thehomopolymer is conjugated to one functional group of this linker whereasthe N-terminal amino acid as well as the lysine group of the OB proteincan be conjugated to the other functional group of this linker. Tri- orpoly- [polyethylene or polypropylene glycol] polymers, conjugates withthe OB protein are formed by using a tri-functional or poly-functionallinker. The homopolymer can be conjugated to two or more of thesefunctional groups with one remaining functional group of the linkerbeing attached to the OB protein. Among these linkers are thosepoly-functional linkers having amine and carboxy functional groups.Amine groups can conjugate with the functionalized hydroxy group of thepolyethylene or polypropylene glycol to form an amide linkage. Carboxygroups can conjugate with the amine groups on the OB protein to form anamide bond and with the functionalized hydroxy group on the glycol toform an ester. Among the many types of linking groups which can beutilized to form the conjugate between the OB protein and the PEG arethose disclosed in U.S. Pat. No. 4,902,502, Nitecki; U.S. Pat. No.5,034,514, Nitecki; U.S. Pat. No. 4,609,546, Hiratani; U.S. Pat. No.5,122,614, Zalipsky; and U.S. Pat. No. 4,847,325, Shadle.

In accordance with an especially preferred embodiment of this inventionare those conjugates of the formula ##STR1## where P is the murine orhuman OB protein described herein; and n and n' are an integers whosesum is from 300 to 1200 so that the average molecular weight of all PEGunits is from 15 to 60 kDa and the total molecular weight of theconjugate is from 30 kDa to 80 kDa; and R and R' are lower alkyl.

The compound of formula I-A and I-B can be prepared from the knownpolymeric materials ##STR2## by condensing them with the murine or humanOB protein of this invention. Any conventional method of reacting anactivated ester with an amine to form an amide can be utilized. In thereaction illustrated above, the exemplified succinimidyl ester is aleaving group causing the amide formation. Where the compound of formulaII-B is utilized to produce the compound of formula I-B, the reactionwith the murine or human OB protein of this invention is carried out inthe same manner described in connection with the conversion of thecompound of formula II-A to the compound of formula I-A. Thesesuccinimidyl ester such as the compound of formula II-A to produceconjugates with proteins are disclosed in Monfardini et al. BioconjugateChem., 6, 62-69 (1995).

In the case of the compound of formula I-A, the sum of n and n' are from300 to 1500 so as to produce a conjugate having a total averagemolecular weight of PEG units of from 15 to 60 kDa and preferably from35 to 45 kDa. In the preferred embodiment of formula I-A the sum of nand n' is from about 800 to 1200 with the average sum of n and n' beingfrom 850 to 1000. Generally, the preferred ratio of n to n' in thecompounds of formula I-A and II-A is from 0.5 to 1.5 with from 0.8 to1.2 being preferred. In the case of the compound of formula I-B, n ispreferably between 300 to 1500 to produce a compound having from 300 to1500 PEG units with a total molecular weight of from 15 to 60 kDa andpreferably from 35 to 45 kDa. In the preferred embodiment n is fromabout 850 to 1000.

Pharmaceutical Compositions

The human or murine ob proteins prepared in accordance with thisinvention may be prepared in pharmaceutical compositions suitable forinjection with a suitable carrier or vehicle by methods known in theart. Among the preferred carriers for formulating the homogeneous OBproteins of the invention are human serum albumen, human plasmaproteins, etc.

Treating Obesity and Related Conditions and Diseases

Administration of recombinant homogeneous ob protein, be it human ormurine or a combination thereof, results in decreased food intake andweight loss in obese humans and animals. Therefore, administration ofthe ob protein replenishes this protein which is important in theregulation of body weight. The pharmaceutical compositions containingthe human or murine ob proteins may be formulated at a strengtheffective for administration by various means to a human or animalpatient experiencing abnormal fluctuations in body weight, either aloneor as part of an adverse medical condition or disease, such as type IIdiabetes mellitus. A variety of administrative techniques by injectionmay be utilized, among them subcutaneous, intravenous andintraperitoneal injections. Average quantities of the ob protein mayvary and in particular should be based upon the recommendations andprescription of a qualified physician or veterinarian.

EXAMPLES

The Examples provided below are not intended to limit the invention isany way.

Example 1

Obtaining the Human ob cDNA

The human ob cDNA was obtained by screening a commercially availablelambda phage cDNA library ("Clontech") made from RNA derived from humanadipocyte tissue. From this library, two lambda phages each containingapproximately a 2.5 kilobase fragments corresponded to the human ob cDNAsequence were obtained through hybridization of lambda phage libraries.By this technique, two clones were identified (i.e., hob1 cDNA and hob2cDNA). The human ob gene was subcloned into the commercially availableplasmid vector DNA (pBluescriptSk⁻ by "Stratagene"). The resultingvectors containing these human ob gene sequences were calledpBluescriptSk⁻ hob1 and pBluescriptSk⁻ hob2.

The human ob gene sequence in this pBluescriptSk⁻ hob1 andpBluescriptSk⁻ hob2 were verified by nucleotide sequencing. The aminoacid sequences of the protein deduced from the nucleotide sequencingcorresponded to the human ob protein of SEQ ID. NO. 4 and as publishedby Zhang, et al (1994). The pBluscriptSk⁻ hob1 had a T-C mutation afterthe stop codon of the hob1 cDNA. This mutation resulted in the loss ofthe StuI restriction site otherwise predicted to be present in thenucleotide sequence of hob 1 as follows: hob 1 - ...GGG.TGC.TGA GGCCTTGA...(SEQ ID NO:9) - Gly Cys stop - pBluscriptSk⁻ hobl - ...GGG.TGC.TGAGGCCC TGA...(SEQ ID NO:10) - Gly Cys stop - pBluscriptSk⁻ hob2 -...GGG.TGC.TGA GGCCT TGA(SEQ ID NO:9) - Gly Cys stop

Since this mutation in pBluescriptSk⁻ hob1 is located after the stopcodon of the human ob cDNA sequence it does not lead to a change in theamino acid sequence of the human ob protein as published by zhang et al.

As far as the nucleotide sequence of the cDNA present in pBluescriptSk⁻hob2 is concerned, it was demonstrated by restriction enzyme analysisthat this plasmid has the StuI restriction site located after the stopcodon of the human ob cDNA sequence.

In addition to the fact that pBluescriptSk⁻ hob1 has a mutation in theStuI restriction site following the stop codon, the pBluescriptSk⁻ hob1also has an EcoRI restriction site after the ORF in hob1 cDNA which isabsent in the hob2 cDNA. (See FIG. 1.)

Example 2

Plasmid Construction for Murine Ob Protein (mob)

Murine ob cDNA of SEQ ID NO. 1 was obtained by the procedure of Zhang etal (1994) and thereafter inserted into the pCDNA3 vector. The murine obgene thus obtained was used to construct the expression vectorpLPPsOmpA-mob for expression of the murine ob protein (mob). Thisexpression vector and its construction is detailed in FIG. 2.

The first stage of construction was to achieve the fusion of thesignal-coding sequence from sOmpA gene to the mature coding region ofthe murine ob gene, i.e., without its natural signal-sequence. The DNAfragment of 501 bp encoding the mature murine ob protein inserted in thepCDNA3 vector was amplified from the vector by the polymerase chainreaction (PCR) using Vent DNA polymerase (New England Biolabs), aforward primer (primer 1) starting with the first nucleotide of thecodon encoding valine (which is the first amino acid in the mature mob)(Zhang et al., Nature 372:425 (1994)), and a reverse primer (primer 2)corresponding to the region of the mob containing the stop codon of mob.Primer 2 also contained a sequence corresponding to a Hind IIIrestriction site.

    PRIMER 1:                                                                       5' GTG CCT ATC CAG AAA GTC 3'(SEQ ID NO:11)                                      Val Pro Ile Glu Lys Val(SEQ ID NO:12)                                       - PRIMER 2:                                                                  5' TCCCAAGCTT TCAGCATTCAGGGCTAAC 3'(SEQ ID NO:13)                                   HindIII stop                                                      

The amplified 501 bp DNA fragment was purified by agarose gelelectrophoresis and phosphorylated using T4 polynucleotide kinase("Boehringer") and next digested with the restriction enzyme HindIII tocreate a 5' protruding end at the position of the primer 2. The obtainedfragment had a blunt end corresponding to the first nucleotide of thecDNA encoding mature mob, and a 5' protruding end corresponding to acleaved HindIII site.

Next, the sOmpA plasmid pT10sOmpArPDI obtained by the methods of DeSutter K. et al (1994) was fused to the mob gene to create apT10sOmpAmob plasmid. To carry this out, the mob fragment was cloned byligation using T4 ligase ("New England-Biolabs") into the pT10sOmpArPDIvector DNA which was previously digested with the restriction enzymesNaeI and HindIII by methods known in the art (Sambrook, J. et al(1989)). This pT10sOmpArPDI plasmid was derived from plasmid p714 (agift to Dr. W. Fiers from Dr. D. Wiley, ref. Parker and Wiley, Gene 83,p 117-134, 1989) and was obtained from the Laboratory of MolecularBiology (Dr. W. Fiers, University of Gent). This plasmid contained thecDNA encoding mature rat protein disulfide isomerase (rPDI) cDNA fusedto the sOmpA sequence.

This fusion of the last codon in sOmpA (alanine) to the first codon inthe cDNA of mature rPDI (glycine) created a NaeI restriction site whichafter cleavage with NaeI released the last codon in the sOmpA sequenceand the first codon in the cDNA encoding rPDI sequence as blunt ends.NaeI - 5' ........GCC / GGC ........3' - Ala Gly - sOmpA cDNA / maturerPDI cDNA

A HindIII site exists at the end of the cDNA encoding rPDI. Therefore,further digestion of this plasmid with HindIIII released the major partof the rPDI cDNA and created a 5' protruding end compatible with one ofthe ends of the PCR fragment. The resulting plasmid where the cDNAencoding the rPDI was replaced by the cDNA encoding mature mouse ob wascalled pT10sOmpAmob and is depicted in FIG. 1.

The ligated DNA was introduced in E. coli strain MC1061 using standardelectroporation and the obtained colonies were screened for the presenceof the murine ob DNA fragment by restriction enzyme analysis. ClonepT10sOmpAmob had the sequence encoding the mature murine ob proteinfused to the sequence encoding sOmpA.

Next, the expression of mob in E. coli in this pT10sOmpAmob was placedunder the control of both the lipoprotein promoter (P_(lpp)) and the lacpromoter-operator (PO_(lac)). To do this, the hybrid gene sOmpA-mobsequence was transferred from the plasmid pT10sOmpAmob to the plasmidvector pLPPsOmpArPDI by standard procedures described in De Sutter et al(1994)). The pLPPsOmpArPDI plasmid was derived from plasmid p714 (a giftto Dr. W. Fiers from Dr. D. Wiley, ref. Parker and Wiley, Gene 83, p117-134, 1989) and was obtained from the Laboratory of Molecular Biology(Dr. W. Fiers, University of Gent). For this step, the plasmidpT10sOmpAmob DNA was cleaved with the restriction enzymes XbaI andHindIII. The fragment containing the sOmpA-mob encoding DNA was thenligated into the plasmid pLPPsOmpArPDI from which the sOmpA-rPDIencoding DNA was previously removed by cleavage with the restrictionenzymes XbaI and HindIII. The resulting plasmid was called pLPPsOmpAmob.

Example 3

Expression of murine ob protein in E. coli (MC1061)

Expression of the murine ob protein in E. coli was achieved as follows.The pLPPsOmpAmob plasmid constructed in accordance with Example 2 wasinserted by electroporation into an E. coli strain MC1061. The E. colicells (MC1061) harboring the plasmid pLPPsOmpAmob was grown up overnightat 28° C. in Luria-Bertania ("Difco Laboratories") medium supplementedwith the antibiotic carbenicillin (100 ug/ml, "Beecham"). This culturewas then used as an inoculum (100-fold dilution) for a 30 ml overnightculture at 28° C. in the same medium. This culture was then diluted100-fold in 3 liter (e.g., 6×0.5 1 in 1 liter erlenmeyer flasks) in theabove medium and shaked at 28° C. in a New Brunswick air shaker (300rpm) for about 4 hours until a density of A₆₀₀ 0.3 to 0.5 is reached. Atthis time, the lac promoter was induced by addition of 2 mM finalconcentration of isopropyl-β-D-thiogalactopyranoside (IPTG,"Boehringer") as described in De Sutter, et al (1994). The cells werefurther incubated at 28° C. for about 5 hours until the cell densityreached A₆₀₀ of 1.3 to 1.5. Next the cells were collected bycentrifugation in a JA10 rotor (Beckman centrifuge models J2-21 orJ2-21M) for 6 min. at 6750 rpm (8000×g) at 4° C. The supernatant wasremoved and the cell pellet was resuspended rapidly in 250 ml icecoldosmotic shock buffer (100 mM Tris-HCl, pH 7.4/ 20% sucrose/10 mM EDTA)and incubated on ice for 10 to 20 min as described by Koshland andBotstein (1980).

Thereafter, the suspension was transferred to plastic centrifuge tubesand the cells collected by centrifugation at 8200 rpm (8000×g) for 5min. at 4° C. in a JA20 rotor. The supernatant was removed, and the cellpellet rapidly resuspended in 120 ml icecold water under vigorousshaking and incubated on ice for an additional 10 min. The suspensionwas then centrifuged in the JA20 rotor for 6 min. at 4° C. at 11,500 rpm(16,000×g) and the supernatant corresponding to the periplasmic fraction(osmotic shock fluid) was collected (approx. 120 ml). Sodium azide andTris-HCl (pH 7.5) was added to a final concentration of 0.05% and 50 mMrespectively. The osmotic shock fluid containing the murine ob proteinwas stored at -20° C. until further use.

Example 4

Expression of Murine Ob Protein in E. coli (MC1061)

Expression of murine ob protein was achieved in accordance with theprocedure described in Example 3, except triacilline (100 μg/ml) was theantibiotic used to supplement the Luria-Bertaria medium (rather thancarbenicillin).

Example 5

Purification of Murine Ob Protein from the E. coli Osmotic Fluid

The murine ob protein located in the 120 ml. frozen osmotic shock fluidin accordance with Example 4 was purified as follows. The 120 ml osmoticshock fluid containing the murine ob protein was thawed and centrifugedat 4° C. for 20 min at 16,000 rpm in a JA20 rotor to remove insolubledebris. The supernatant was then loaded directly onto a columncontaining a 30 ml bedvolume Q-Sepharose Fast Flow ("Pharmacia")preequilibrated with 50 mM Tris-HCl (pH 7.5) buffer. After washing withthe 50 mM Tris-HCl (pH 7.5) buffer, the mob protein was eluted with 50mM Tris-HCl (pH 7.5) buffer containing 0.1 M NaCl.

Next, solid (NH₄)₂ S0₄ was added to the material eluted from theQ-Sepharose Fast Flow containing column to a final concentration of 1.0M and the mixture was loaded onto a column containing 7.5 ml bedvolumeButyl-Sepharose Fast Flow ("Pharmacia") preequilibrated with 50 mMTris-HCl (pH 7.5) buffer containing 1.0 M (NH₄)₂ S0₄. After washing withTris-HCl (pH 7.5) buffer containing 1.0 M (NH₄)₂ S0₄, the mob proteinwas eluted with by applying a gradient from 1.0 M (NH₄)₂ SO₄ in 50 mMTris-HCl (pH 7.5) buffer to 20% ethylene glycol in water. The mobprotein eluted from the Butyl-Sepharose Fast Flow column at the very endof the gradient, while most contaminants eluted much earlier. The purityof the mob protein at this stage was 90% as estimated by silver-stainedpolyacrylamide gel electrophoresis (PAGE).

The mob protein in the material eluted from the Butyl-Sepharose FastFlow containing column was then further purified by gel filtrationchromatography. To do this, mouse ob protein was concentrated at 4° C.to a volume of 1 ml on a YM10 ("Amicon") membrane using a 8MCconcentrating unit ("Amicon"), and was applied to a column (1.0 cm×50cm) containing 39 ml GlOO-Sephadex ("Pharmacia") preequilibrated inphosphate buffered saline. The fractions containing the mob protein werethen pooled and the protein concentrated on a YM10 membrane. At thisstage, the mob protein was more than 95% pure as estimated by PAGE andsilver staining. SDS-PAGE revealed a single protein band at Mr 15,000.

Example 6

Sequence Analysis of Murine Ob Protein

N terminal amino acid sequence of the murine ob protein obtained andpurified by the procedures of Examples 2, 3, 4 and 5 described above wasperformed according to the procedure of Laemli, U. K. (1970). Afterelectrotransfer of the electrophoresed proteins to a poly(4-vinylN-methylpyridinium iodide)-coated glass fiber sheet as described by Bauwet al (1988), the band of protein with Mr 15,000 was excised from themembrane and the N-terminal amino acid sequence was determined by Edmandegradation on a 470A gas-phase sequenator equipped with a 120A on-linephenylthiohydantoin amino acid analyzer ("Applied Biosystems"). TheN-terminal amino acid sequence of the murine ob protein was obtained bythe above described procedures was Val-Pro-Ile-Gln corresponding to themature murine ob protein of SEQ ID NO: 3.

Example 7

Construction of Expression Vector for Human Ob Protein (hob)

The human ob gene obtained in accordance with the procedures of Example1 was utilized to construct an expression vector pLPPsOmpA-hob1 forexpression of the human ob protein. This construction was similar to theconstruction of pLPPsOmpA-mob described in Example 2 and is detailed inFIG. 3. A three-fragment ligation was required to complete the DNAfragment containing the entire mature human ob coding sequence.

In the first stage of the construct, a hob nucleotide sequence startingwith the first nucleotide of the codon encoding the first amino acid ofthe mature human ob protein (valine) was fused to the signal-codingsequence from OmpA (sOmpA), so that the sOmpA sequence is upstream ofthe 5' end of the hob nucleotide coding sequence. This DNA fragment wasobtained by amplification in a PCR mixture containing plasmidpBluescriptSk⁻ hob1 , Vent DNA polymerase, and two primers. The forwardprimer (primer 1) started with the first nucleotide of the codonencoding the first amino acid of mature human ob protein, and thereverse primer (primer 2) contained the sequence of the human cDNAcontaining the stop codon. The amplification reaction yielded a DNAfragment of 501 bp containing the sequence encoding the mature human obprotein. The 5' end of this DNA fragment was then phosphorylated with T4polynucleotide kinase and digested with the restricition enzyme HindIII,yielding a 353 bp DNA fragment having a blunt end corresponding to thefirst nucleotide of the cDNA encoding mature hob (position correspondingto the primer 1), and a 5' protruding end corresponding to a cleavedHindIIII site. This 353 bp DNA fragment was purified by agarose gelelectrophoresis and cloned in the pT10sOmpArPDI plasmid which has beenpreviously digested with the restriction enzymes Nae I and HindIII. Theresulting plasmid pT10sOmpAhob1 -partial, has the DNA fragment encodinga part of the mature human ob protein (amino-terminal part) fused to thesequence encoding sOmpA. Primer 1: 5' GTGCCCATCCAAAAAGTC 3'(SEQ IDNO:14) - Primer 2: 5' TCCCAAGCTTTCAGCACCCAGGGCTGAG 3'(SEQ ID NO:15) -stop

In a second step, the DNA sequence encoding the carboxy terminal part ofthe human ob protein (i.e., fragment 2) was ligated to the DNA fragmentencoding the amino-terminal part of mature hob, and the resultingfragment encoding the entire mature hob sequence fused to the sOmpA wastransferred to plasmid pLPPsOmpArPDI to bring expression of mature humanob protein in E.coli under the control of the lipoprotein promoter andthe lacpromoter-operator. To do this, the plasmid pT10sOmpAhob1 -partialwas digested with Xbal and HindIII and the 400 bp DNA fragment 1 of hobwas isolated by agarose gel electrophoresis (fragment 1). Next theplasmid pBluescriptSk⁻ hob1 was cleaved with HindIII and EcoR1, and the450 bp was isolated by agarose gel electrophoresis (fragment 2).Finally, the plasmid pLPPsOmpArPDI was cleaved with XbaI and EcoRI, andthe vector fragment isolated by agarose gel electrophoresis (fragment3). The DNA fragments 1, 2 and 3 were then ligated to each other and theligation mixture introduced into E. coli strain MC1061. The colonycontaining the final plasmid construct pLpPPsOmpAhob1 was used forexpression and secretion of mature human ob protein.

Example 8

Expression of Human Ob Protein in E. coli (MC1061)

The pLPPSOmpAhob1 constructed in accordance with Example 7 was used totransform E. coli strain MC1061 for expression of the human ob proteinin soluble biologically active form in the periplasm of the host E. colicells. Insertion of the plasmid into these host E. coli cells wasperformed by electroporation. The E. coli cells (MC1061) harboring theplasmid pLPPsompAhob1 were grown at 28° C. in Luria-Bertania ("DifcoLaboratories") medium supplemented with the antibiotic carbencillin (100ug/ml, "Beecham") to the proper density, after which the lac promoterwas induced by addition of 2 mM final concentration ofisopropyl-β-D-thiogalactopyranoside (IPTG, "Boehringer") as described inDe Sutter, et al (1994). The cells were further grown until the celldensity reached 1.3 A₆₀₀. Next the cells were collected bycentrifugation (8000×g at 4° C. ) and the cell pellet was resuspendedrapidly in icecold osmotic shock buffer (100 mM Tris-HCl, pH 7.4/20%sucrose/10 mM EDTA) and incubated on ice for 10 min as described byKoshland and Botstein (1980).

Thereafter, the cells were again collected by centrifugation as aboveand the cell pellet was resuspended in ice cold water and incubated onice for 10 min. The suspension was then centrifuged for 5 min. at16,000×g and the supernatant (osmotic shock fluid) was collected. Sodiumazide and Tris-HCl (pH 7.5) was added to a final concentration of 0.05%and 50 mM respectively. The osmotic shock fluid containing the human obprotein was stored at -20° C. until further use.

Example 9

Expression of Human Ob Protein in E. coli (Mc1061)

Expression of the human ob protein was achieved by using the procedureof Example 8, except triacilline (100 μg/ml) was used as the antibioticto supplement the Luria-Bertaria medium rather than carbenicillin.

Example 10

Purification of Human Ob Protein from the E. coli osmotic Fluid

To purify the human ob protein in the osmotic shock fluid of Example 9,NaCl was added to the fluid to a final concentration of 0.1 M and thefluid was then loaded directly onto a column containing a 30 mlbedvolume Q-Sepharose Fast Flow ("Pharmacia") preequilibrated with 50 mMTris-HCl (pH 7.5) buffer.

Next, solid (NH₄)₂ S0₄ was added to the flow-through material elutedfrom the Q-Sepharose Fast Flow containing column to a finalconcentration of 1.0 M and the mixture was loaded onto a columncontaining 7.5 ml bedvolume Butyl-Sepharose Fast Flow ("Pharmacia")preequilibrated with 50 mM Tris-HCl (pH 7.5) buffer containing 1.0 M(NH₄)₂ SO₄. After washing with Tris-HCl (pH 7.5) buffer containing 1.0 M(NH₄)₂ SO₄, the hob protein was eluted with by applying a gradient from1.0 M (NH4)2SO4 in 50 mM Tris-HCl (pH 7.5) buffer to 20% ethylene glycolin water. The hob protein eluted from the Butyl-Sepharose Fast Flowcolumn at the very end of the gradient, while most contaminants elutemuch earlier. The purity of the hob protein at this stage was 90% asestimated by silver-stained polyacrylamide gel electrophoresis (PAGE).

The hob protein in the material eluted from the Butyl-Sepharose FastFlow containing column was then further purified by gel filtrationchromatography. To do this, human ob protein was concentrated at 4° C.to a volume of 1 ml on a YM10 ("Amicon") membrane using a 8MCconcentrating unit ("Amicon"), and was applied to a column (1.0 cm×50cm) containing 39 ml G100-Sephadex ("Pharmacia") preequilibrated inphosphate buffered saline. The fractions containing the hob protein werethen pooled and the protein was concentrated on a YM10 membrane. At thisstage, the hob protein was more than 95% pure as estimated by PAGE andsilver staining. SDS PAGE analysis of the eluate revealed a singleprotein band at Mr 15,000.

Example 11

Purification of Human Ob Protein from the E. coli Osmotic Fluid

To purify the human protein in the osmotic shock fluid of Example 9 theprocedure of Example 10 was used with the following exception: Prior toadding solid (NH4)₂ SO₄ to the flow-through material, the followingsteps were carried out with regard to the osmatic shock fluid of Example9.

The human ob protein in the osmotic shock fluid of Example 9 was loadeddirectly onto a column containing a 30 ml bedvolume Q-Sepharose FastFlow ("Pharmacia") prequilibrated with 50 mM Tris-HCl (ph 7.5) buffer.After washing with the 50 mM Tris-HCl (pH 7.5) buffer, the hob proteinwas eluted with 50 mM Tris-HCl (pH 7.5) buffer containing 0.1 M NaCl.

Example 12

Sequence Analysis of Human Ob Protein

N terminal amino acid sequence of the human ob protein purified andobtained by the procedures of Examples 7-11 described above wasperformed according to the procedure of Laemli, U. K. (1970). Afterelectrotransfer of the electrophoresed proteins to a poly(4-vinylN-methypyridinium iodide)-coated glass fiber sheet as described by Bauwet al (1988), the band of protein with Mr 15,000 was excised from themembrane and the N-terminal amino acid sequence was then determined byEdman degradation on a 470A gas-phase sequenator equipped with a 120Aon-line phenylthiohydantoin amino acid analyzer ("Applied Biosystems").The N-terminal amino acid sequence of the hob protein obtained by theabove described procedures was Val-Pro-Ile-Gln corresponding to themature human ob protein of SEQ ID NO: 6.

Example 13

Biological Activity of Murine Ob Protein: Intracerebroventricular (ICV)Injection in ob/ob Mice

The biological activity of the mature murine ob protein purified inaccordance with Example 5 was determined using the ICV method asfollows. Infusion cannulas were implanted into the lateral ventricle ofthe brains of anesthetized female obese ob/ob mice (age 6-13 weeks)using the following coordinates (2 mm lateral of midline; 0.6 mm withrespect to bregma; 2 mm down) based on the methods of Haley and McCormick (1957). The end of the cannula was mounted on the skull using ajeweler screw and dental cement. Mice were individually housed inplastic cages with free access to food (except for the night prior toICV injection) and water. Following recovery from surgery as assessed bydaily food intake and body weight gain, mice were studied on severaloccasions following the intracerebroventricular (ICV) injection of 1 ulof one of the following test solutions:

1) artificial CSF;

2) bacterial control solution;

3) ob protein (0.6 to 1 ug/mouse); or

4) no infusion.

The injection of one of the above test solutions ICV into each mice wasfollowed by 1 ul of artificial CSF to clear the cannula. For purposes ofthis experiment, the bacterial control solution was an sampleidentically processed and prepared in accordance with the proceduresoutlined for Examples 2-4, except that the plasmid inserted in the E.coli bacteria was absent the murine ob gene.

Mice were fasted for 18 hours (overnight) prior to ICV injection. Micewere lightly restrained and a 10 ul Hamilton syringe fitted with a pieceof precalibrated polyethylene (PE) tubing (PE20) was used to inject lulof the test solution into the cannula placed in the lateral ventricle.Mice were then immediately placed in a test cage with a food dishcontaining a pre-weighted amount of pelleted mouse chow and a waterbottle. Mice were visually observed and food intake was measured for thenext seven hours. Food intake measurements were obtained at 0.5, 1, 2,3, 4, 6 and 7 hours post-ICV injection. Body weight for each animal wasmeasured prior to the ICV injection and 24 hours later. Successfulcannula placement was documented by an increase in 2 hour food intakefollowing ICV injection of 10 ug Neuropeptide Y in 2 hour fasted miceaccording to Morley, J. E. et al (1987).

The results of the ICV test described above were as follows:

A. Reduction of Food Intake

During the first 30 minutes following ICV injection almost all mice atewith a short latency and consumed approximately 0.5 grams. Mice whichreceived no injection or artificial CSF continued to eat throughout thenext 6.5 hours and reached a cumulative 7 hour intake of 3.2 grams(Table 1). In contrast, the mice treated with ob protein ICV stoppedeating after the first 30 min and did not eat again. Thus, theircumulative food intake remained suppressed over the next 6.5 hours atapproximately 0.5 grams (Table 1). Mice receiving the vehicle controlsolution ICV also stopped eating after 30 min., and only began eatingagain between 6 and 7 hours.

B. Reduction in Body Weight Gain

The 24 hour change in body weight of the mice injected with vehiclecontrol was slightly reduced from that of artificial CSF injected ornon-injected mice (Table 1). However, the percent change in body weightof the mice injected with ob protein was near zero and was significantlyreduced compared to the vehicle control injected mice (Table 1).

C. Conclusion

The observed effect of direct administration of recombinant mouse obprotein (1.1 ug/mouse in 1 ul to the brain) was a sustained andsignificant reduction in food intake and body weight gain of femaleob/ob mice. This demonstrates that ob protein can act directly on thebrain and is consistent with the effect of the ob protein when injectedintraperitoneal. This example also confirms the biological activity ofbacterially expressed recombinant murine ob protein in female obeseob/ob mice in accordance with the invention.

                  TABLE 1                                                         ______________________________________                                                Food Intake       Body Weight Gain                                      (0.7 hr)  (0-24 hr)                                                         Treatments                                                                              g         %**       g       %                                       ______________________________________                                        Artifical CSF                                                                           3.2 ± 0.2                                                                            100       3.8 ± 0.3                                                                          100                                       Vehicle Control 0.9 ± 0.3 28.1 2.9 ± 0.2  76                            Ob Protein  0.5 ± 0.1* 15.6  0.3 ± 0.5*  8                              (1 μg/mouse)                                                             ______________________________________                                         *indicates significant differences between ob protein and artificial CSF      groups with p < 0.05.                                                         **indicates percent of control                                           

Example 14

Biological Activity of Murine Ob Protein Intravenous (IV) in Ob/Ob Mice

The biological activity of the murine ob protein obtained and purifiedin accordance with Examples 2, 3, 4 and 5 was tested by intravenous (IV)injection in obese ob/ob mice as follow.

Male and female obese ob/ob mice (6-13 weeks old) were implanted withchronic jugular cannulas under pentobarbital anesthesia (80 mg/kg bodyweight) according to the method of Mokhtarian A., et al. (1993). Micewere individually housed in plastic cages under constant environmentconditions with a 12 hr dark/12 hr light cycle. Body weights weremeasured in fusion daily and the patency of cannulas was verified andmaintained every other day by infusion of <0.1 ml sterileheeparin/saline solution (50 U/ml in 0.9% saline). After completerecovery from surgery, assessed by body weight gain, the mice werefasted 16-18 hours (overnight). The next morning, mice were weighed andplaced in test cages for 45 minutes for acclimatization before theexperiment. Water was available contintously. Mouse ob protein (3 ug in0.1 ml) or an equal volume of vehicle control or saline (0.9%) solutionwas injected intravenously. Awake mice were lightly restrained and 0.5ml insulin syringes were used to inject 0.1 ml of the test solutionfollowed by 0.05 ml heparin/saline. Trials were separated by at least 3days. Mice were then immediately replaced in the test cage with apre-weighted petri dish containing a pellet of mouse chow. Mice werevisually observed and food intake was measured for the next seven hoursat 0.5, 1, 2, 3, 4, 6 and 7 hrs post-IV injection. Body weight wasmeasured before the IV injection and again 24 hrs later. Two separategroups of cannulated mice (11 ob/ob and 12 lean) were used in fiveindividual trials. Most mice received mouse ob protein and one or bothcontrol injections in counterbalanced order. Two separate preparationsof mouse ob protein were used in this experiment. The data reported hereare a combination of the results of these individual replications.

A. Results

The results of the above experiment are as follows:

During the first 30 minutes following IV injection most obese and leanmice ate with a short latency and consumed approximately 0.3-0.5 grams.Food intake in saline and vehicle control injected obese mice increasedthroughout the experiment. The cumulative food intake of obese ob/obmice injected with vehicle control was not different from the foodintake of similarly fasted obese mice that were injected with saline. Incontrast, the food intake of the obese ob/ob mice injected withrecombinant ob protein was significantly reduced and remained suppressedat 57% of control (Table 2). No other behavioral effects were observedin the vehicle control and ob protein groups throughout the 7 hrobservation period. As expected, the 24 hr post-injection body weightgain was not different in the treatment groups (Table 2) due to thelimited duration of action of a single IV bolus of mouse ob protein.

B. Conclusions

These results demonstrate that recombinant mouse ob proteinsignificantly reduced cumulative 7 hr food intake following IVadministration (3 μg/mouse) in obese ob/ob mice. The ability ofrecombinant mouse ob protein to reduce food intake in obese ob/ob miceis consistent with the food intake results obtained following repeatedIP injection of the ob protein in obese ob/ob mice. This example alsoconfirms the biological activity of bacterially expressed recombinantmouse ob protein in female obese ob/ob mice.

                  TABLE 2                                                         ______________________________________                                        IV Administration of Murine Ob Protein in Ob/Ob Mice                                     Food Intake        Body Weight Gain                                  (0-7 hr)  (0-24 hr)                                                         Treatments g         %**      g       %                                       ______________________________________                                        Saline     1.8 ± 0.2       2.9 ± 0.3                                      (n = 4)                                                                       Vehicle Control 1.4 ± 0.3 100 2.2 ± 0.6 100                             (n = 7)                                                                       Ob Protein  0.8 ± 0.2*  57 1.4 ± 0.6  64                                (1 μg/mouse)                                                               (n = 8)                                                                     ______________________________________                                         Data are mean ± sem.                                                       n = indicates the number of individual mice.                                  *indicates significant differences between ob protein and artificial CSF      groups with p < 0.05.                                                         **indicates percent of control                                           

Example 15

Biological Activity of the Murine Ob Protein: Repeated IP Injection inOb/Ob Mice.

The biological activity of the murine ob protein obtained and purifiedin accordance with Examples 2-5 was tested by repeated intraperitoneal(IP) injection in obese ob/ob mice as follows.

Three groups of six female obese ob/ob mice were studied. Mice werehoused in plastic cages (three per cage) under constant environmentalconditions with a 12 hour dark/12 hour light cycle. Twenty-four (24)hour food intake and body weight were measured every day. Following anadaptation period to environmental conditions and daily handling andinjections, the mice were sorted into three treatment groups. Each mousereceived two intraperitoneal (IP) injections each treatment day (shortlybefore the beginning of the dark phase of the dark/light cycle and threehours into the dark phase) of the 0.1 ml of the following testsolutions:

1) saline (0.9%);

2) bacterial control solution; or

3) murine ob protein (3 μg/0.1 ml).

The bacterial control solution was a sample identically processed andprepared in accordance with the procedures outlined for Examples 2-4 toobtain and purify murine ob protein, except that the plasmid inserted inthe E. coli bacteria was absent the murine ob gene. Mice were treatedtwice daily for five days and then received no treatment for two days.Food intake of each cage was measured at 2, 3, 5 and 24 hours followingthe first IP injection on each treatment day.

The results of the above experiment are as follows:

A. Reduction of Food Intake

Food intake was not different in the saline and bacterial controlinjected mice on treatment and non-treatment days throughout the oneweek experiment (Table 3). However, food intake was reduced in the sixmice injected with 6 ug ob protein on treatment days throughout theexperiment. The reduction in food intake was observed at 2, 3, 5 and 24hours after the first injection on treatment days in the mice receivingob protein group compared to the bacterial control and saline controlgroups.

B. Reduction of Body Weight Gain The cumulative body weight gain overthe five treatment days of the ob protein group was -3.3±0.7 gramscompared to -0.9±0.2 grams in the saline and -0.7±0.4 grams in thebacterial control groups (Table 3).

C. Conclusion

This example demonstrates that two daily IP injections of bacteriallyexpressed recombinant murine ob protein (6 ug/mouse/day) resulted in asignificant, sustained reduction of food intake and a significantdecrease in the rate of weight gain of treated female ob/ob micecompared to saline and bacterial control treated ob/ob mice. Theseresults demonstrate the bacterial expressed murine ob protein isbiologically active and has the expected anti-obesity effects ongenetically obese ob/ob mice in accordance with this invention. In Table3 the 2 and 5 hour results are the mean daily food intake while the 24hour results shown are the 5 day cumulative food intake.

                                      TABLE 3                                     __________________________________________________________________________    Repeated IP Administration of Murine Ob Protein in Ob/Ob Mice                       Food Intake                                                               (grams/3 mice) Body                                                         2 hr            5 hr      24 hr     Weight                                                % of      % of      % of                                                                              Gain                                        Treatment g control g control g control grams                               __________________________________________________________________________    No Injection                                                                        5.5 ± 0.5                                                                            14.5 ± 0.5                                                                           44.3 ± 3.5                                                                           -0.9 ± 0.2                               Control 7.0 ± 0.5 100 16.5 ± 1.5 100 49.5 ± 6.1 100 -0.7 ±                                          0.4                                         Ob Protein  3.5 ± 0.5*  50  5.5 ± 1.0*  33  25.5 ± 4.6*  52                                            -3.3 ± 0.7*                            __________________________________________________________________________     Data are mean ± sem for six ob/ob mice in each group.                      Food intake is mean cumulative intake for cages of three mice during the      five days of treatment at 2, 5 and 24 hr after the first IP injection.        Body weight gain is the cumulative change in body weight during the five      days of treatment.                                                            *indicates significant differences between ob protein and vehicle groups      with p < 0.05.                                                           

Example 16

Biological Activity of Human Ob Protein: Intracerebro Ventricatar (ICV)Injection in Ob/Ob Mice

The methods used to determine biological activity of Human Ob protein byintracerebroventricular ICV injection in Ob/Ob Mice were the same asExample 13 except that the test solutions were:

Recombinant human ob protein produced in Example 11 (0.05 μg) inphosphate buffered saline (PBS) containing 0.1% mouse serum albumin; and

PBS containing 0.1% (w/v) mouse serum albumin (albumin control) as thevehicle control solution.

A. Reduction of Food Intake

During the first 30 minutes following ICV injection almost all mice atewith short latency and consumed approximately 0.5 grams. Mice whichreceived no injection continued to eat throughout the next 6.5 hours andreached a comulative 7 hour intake of 1.8 grams (Table 4). Micereceiving the albumin control solution ICV also stopped eating after 30minutes and only began eating again between 3 and 7 hours. In contrast,the mice treated with human ob protein ICV ate significantly less in thefirst 30 minutes (0.2 grams) and ate very small amounts during the next6.5 hours. Thus, their cumulative food intake remained suppressed overthe next 6.5 hours at approximately 0.4 grams (Table 4).

B. Reduction in Body Weight Gain

The 24 hour change in body weight of the mice injected with vehiclecontrol was slightly reduced from that of artificial CSF injected ornon-injected mice (Table 4). However, the percent change in body weightof the mice injected with human ob protein was near zero (Table 4).

C. Conclusion

The observed effect of direct administration of recombinant human obprotein (0.05 μg/mouse in 1 μl) to the brain to lead to a substained andsignificant reduction in food intake and body weight gain of femaleob/ob mice demonstrates that ob protein can act directly on the brainand is consistent with the effect of ob protein when injected IP. Thisexample also confirms the biological activity of bacterially expressedrecombinant human ob protein in female obese ob/ob mice.

                  TABLE 4                                                         ______________________________________                                        ICV Administration of Human Ob Protein in Ob/Ob Mice                                      Food Intake        Body Weight Gain                                 (0-7 hr)  (0-24 hr)                                                         Treatments  g         %**      g      %**                                     ______________________________________                                        No Injection                                                                              1.8 ± 0.2       3.1 ± 0.4                                     (n = 3)                                                                       Albumin Control 1.0 ± 0.4 100 1.7 ± 0.6 100                             (n = 3)                                                                       Human Ob Protein  0.4 ± 0.2*  40   0 ± 0.8  0                           (1 μg/mouse)                                                               (n = 5)                                                                     ______________________________________                                         Data are mean ± sem.                                                       n = indicates the number of individual mice.                                  *indicates significant differences between human ob protein and artificia     CSF groups with p < 0.05.                                                     **indicates percent of control                                           

Example 17

Biological Activity of Ob Protein in Obese Human Subjects: Reduction ofTest Meal Intake Following IV Administration

The biological activity of the murine and human ob proteins obtained andpurified in accordance with Examples 7-11 respectively, is determined bymeasuring test meal intake following IV administration to humans asfollows.

Lean and obese human volunteers are presented with test meals of fixedcaloric content in an eating laboratory on two occasions using themethod of Muurahainen, N. E. et al (1991). At least one hour prior tomeal presentation, an indwelling IV catheter is placed in theantecubital or forearm vein and is kept open with a heparin lock.Visual-analog hunger rating are obtained 15 minutes before, 15 minutesafter meal presentation, and at the conclusion of the test meal. Murineor human ob protein or saline is then infused IV 20 minutes prior tomeal presentation. Each subject is instructed to eat as much of the testmeal as they wish until they are satisfied. The amount of the test mealingested by each subject is measured. Each subject then receivesinfusions of either human ob protein (0.5 mg/kg body weight), murine obprotein (0.5 mg/kg body weight) or saline and the difference in amountof the test meal ingested under these conditions is calculated. In thehuman or murine ob protein group, there is a reduced amount of mealconsumed by at least 20%.

Example 18

Biological Activity of Ob Protein in Obese Human Subjects: Induction ofWeight Loss by Repeated IV Administration

The biological activity of the murine and human ob proteins obtained andpurified in accordance with Examples 7-11, respectively, is determinedby measuring weight loss following repeated IV administration of the obprotein, according to the following method.

A placebo controlled, double blind weight loss study using the methodsof Drent, M. L. et al (1995) is performed. Obese subjects (BMI greaterthan 27) are weighed and then placed on a diet with 1500 Kcal for a 2-4week run-in period at the end of the run-in period, all obese subjectsthat lost at least 1 kg body weight are randomized into two treatmentgroups matched for weight loss during the run-in phase. Subjects receivedaily IV administration of either human or murine ob protein (0.5mg/kg/day) or placebo (saline) for at least 6 weeks. body weigh isrecorded weekly. Those subjects receiving human or murine ob proteinhave a significant reduction in body weight than the placebo group after6 weeks of treatment.

Example 19

A. Preparation of Polyethylene Glycol Conjugated OB Protein From E. coliCells

50 g. of E. coli cell pellet prepared in Example 8 prior to resuspensionwas suspended with 1L of 50 mM Tris-HCl (pH 8.5) containing 50 mM EDTA.The suspension was incubated for 15 minutes at 37° C., diluted with anadditional 1L of 50 mM Tris-HCl (pH 8.5) containing 5 mM EDTA.Thereafter, the suspension was homogenized using a homogenizer for 15minutes at 50% power setting. The suspension was clarified bycentrifugation at 8,000 rpm, 4° C. for one hour. The pellet wasdiscarded. The supernatant was diluted with water to a conductivity of1.8 mS, and applied directly onto a column packed with 200 ml ofQ-Sepharose Fast Flow (strong anionic ion exchange resin),preequilibration with 50 mM Tris-HCl (pH 8.5). After washing with theequilibration buffer, the adsorbed OB protein was eluted fran the columnwith the same equilibration buffer which additionally contained 100 mMNaCl. The eluate obtained after treating the column with theequilibration buffer contained sodium chloride was called Q-SepharoseEluate.

Solid NaCl was added to the Q-Sepahrose Eluate to reach the finalconductivity to 82 mS. After this, the eluate was applied onto aHydrophobic Interaction Column (HIC) packed with 200 ml butyl-SepharoseFast Flow, preequilibrated with 50 mM Tris-HCl (pH 8.5) containing 1MNaCl. The unadsorbed materials were washed away with equilibrationbuffer and the adsorbed OB protein was eluted with 50 mM ammoniumacetate (pH 6.9) to produce HIC eluate. The OB protein in the HIC eluatewas determined to be 95% pure by reverse phase HPLC. The purified OBprotein was concentrated to 3.7 mg/ml using a sizing membrane (YM-10).The sizing membrane was a membrane which retained molecules of 10,000daltons or greater. After this concentration step, by using a sizingmembrane, the OB protein was diafiltered into 100 mM borate buffer (pH8.0) which was used as the OB stock solution.

B. Pegylation of Human OB Protein

In carrying out this pegylation reaction, the PEG₂ -NHS reagent offormula II-A wherein R is CH₃, the sum of n and n' range from 820 to1040 with the average sum being about 930 and having an averagemolecular weight of 40 kDa which was purchased from Shearwater Polymers,Huntsville, Alabama was utilized. This was a mixture of PEG₂ -NHSreagents of formula II-A where the ratio of n to n' is approximately 1.0and the sum of n and n' in this mixture ranged from 820 to 1040 unitswith the average molecular weight of the PEG chain in this mixture beingapproximately 20 kDA so that the average molecular weight of the reagentis approximately 40 kDA with the average sum of n and n' in this mixturebeing about 930. To 2 mg or 0.54 ml of the 3.7 mg/ml purified human OBstock solution prepared above in part A [125 nmoles OB protein], therewas added 250 n moles of the aforementioned PEG₂ -NHS reagent solution.This solution consisted of 10 mg or 0.1 ml of the 100 mg/ml PEG₂ -NHSreagent solution in 1 mM HCl. The total reaction mixture was made up to0.67 ml by adding 100M borate pH5.0. Final molar ratio of protein toreagent was 1:2. This mixture was stirred at 4° C. for 4 hours and thereaction was stopped by the addition of 1 microliter of glacial aceticacid to produce a final pH of 4.5. The resulting reacting mixture (0.67mL) was diluted with water to form a 27 ml. solution which was loadedonto a column containing 1.7 ml of carboxy methylated cationic exchangeresin [Popos Perseptives, Framingham, Massachusetts]. The column wasequilibrated with 3.3 μM HEPES/MES/Sodium acetate buffer pH 5.0. Thediluted reaction mixture was applied to the column and unadsorbed PEG₂-NHS reagent was washed off the column. The adsorbed pegylated andunmodified OB proteins were eluted with step salt gradients [15 columnvolumes each] of 80, 150 and 500 mM NaCl. 2ml of these eluates wereseparately collected in sequence and the samples of each fraction weresubjected to an SDS-PAGE analysis. From this analysis, the eluants wereclassified as highly pegylated conjugates, desired branched mono-PEG-OB(PEG₂ -OB) and unmodified OB protein. Each of these fractions werepooled into the classifications set forth above and the second poolcontaining the desired branched mono-PEG₂ -OB protein. This desiredprotein had the structure of compound of formula I-A wherein the sum ofn and n' was approximately 820-1040,with the average sum being about930, R and R' are CH3 and the average molecular weight of each PEG chainwas about 20 kilodaltons. The pegylated product had an average molecularweight of about 56 kDA. The pool containing the PEG₂ -OB wasconcentrated to 3.7 mg/ml using a YM 10 membrane. YM 10 is a sizingmembrane which retains molecules having molecular weight of 10,000daltons or greater. After the sizing step, concentrated material wasdiafiltered into a PBS buffer (pH 7.3) and stored frozen at -20° C. Thisstored product was the pegylated OB protein of Formula I-A where the sumof n and n' was approximately 820 to 1040 and the average molecularweight of each OB chain was approximately 20kDA. The average molecularweight of the PEG protein in this OB protein conjugate mixture was 56kDA.

Example 20

Biological Assay of Pegylated Human OB Protein: Single IP Injection inOb/Ob Mice

Methods

Two groups of six mice female obese ob/ob mice were studied. Mice werehoused in plastic cages (three/cage) under constant environmentalconditions with a 12 hr dark/12 hr light cycle. 24 hr food intake andbody weight were measured every day. Following an adaptation period toenvironmental conditions, daily handling and injections, the mice weresorted into two treatment groups. Each mouse received oneintraperitoneal (IP) injections on day 1 of the experiment (just beforethe beginning of the dark phase of the dark/light cycle) of the 0.1 mlof the following solutions: Saline (0.9%); human OB protein stocksolution prepared in part A of Example 19 (30 μg/0.1 ml); pegylatedcontrol solution (an identically processed and purified sample withouthuman OB protein) or pegylated OB protein (30 μg/0.1 ml) prepared asdescribed in Example 19. Mice were injected only once, on day 1. Dailyfood intake of the cage and the body weight of each mouse was measuredfor the next three days and again on day 6.

Results

A) Reduction of Food Intake

Daily food intake was not different in the saline and pegylation controlinjected mice on the single treatment and two subsequent days (Table 5).However, daily intake was reduced in the six mice injected with 30 μghuman ob protein and pegylated human OB protein on the treatment day(5.2, 8.2 vs 11.9, 11.4 g) compared to the saline and pegylated controlinjected mice. The food intake of the mice injected with human OBprotein returned to control levels, while the food intake of the miceinjected with pegylated human OB protein remained reduced on thesubsequent days of the experiment. The reduction in food intake wasobserved 48 hrs after the single injection in the pegylated human OBprotein group. The cumulative 24 hr food intake over the three days ofthe experiment was significantly reduced to 49% of control in thepegylated human OB protein compared to the saline and pegylation controlgroup.

B) Reduction of Body Weight Gain

Change in body weight was not different in the saline and pegylationcontrol injected mice on the single treatment and two subsequent days(Table 6). However, body weight was reduced in six mice injected with 30μg human Ob protein and pegylated human OB protein on the treatment day(-0.9, -0.7 vs 0.1, 0.3 g) coared to the saline and pegylated controlinjected mice. The body weight of the mice injected with human OBprotein returned to control levels, while the body weight of the miceinjected with pegylated human OB protein continued to decrease on thesubsequent days of the experiment. The continued reduction in bodyweight was observed 48 hrs after the single injection in the pegylatedhuman OB protein group. The cumulative change in body weight over thesix days of the experiment was -1.6 grams compared to 0.4 grams in theOB protein group, 0.7 grams in the saline and 1.1 +0.2 grams pegylationcontrol groups (Table6).

Conclusion

This example demonstrates that a single IP injection of pegylated humanOB protein (30 μg/mouse) resulted in a significant, sustained reductionof food intake and a significant decrease in body weight of treatedfemale ob/ob mice over three days compared to saline and pegylationcontrol treated ob/ob mice. These results demonstrate the pegylatedhuman OB protein has sustained, potent biologically active and has theexpected antiobesity effects on genetically obese ob/ob mice.

                  TABLE 5                                                         ______________________________________                                        Daily Food Intake of a Single IP Administration                                 of Pegylated Human OB Protein in Ob/Ob Mice                                          Food Intake                                                            (/3 mice/day)                                                                            day 1       day 2     day 3                                                       o% f          % of        % of                                 Treatment g control g control g control                                     ______________________________________                                        Saline   11.9    100     13.7  100   13.6  100                                  OB Protein  5.2 43.7 11.7 85.4 12.5 92                                        Pegylated 11.4 95.8  4.5 106 13.4 99                                          Control                                                                       Pegylated OB  8.2 68.9  6.0 43.8  4.9 36                                      Protein                                                                     ______________________________________                                    

Data are mean for six ob/ob mice in each group. Food intake is meandaily food intake for cages of three mice on each day of the experimentafter the single IP injection on day 1. Note persistent reduction indaily food intake only in the pegylated OB protein group.

                  TABLE 6                                                         ______________________________________                                        Change in Body Weight of a single IP Administration                             of Pegylated Human OB Protein in Ob/Ob Mice                                               Change in Body Weight                                             (grams)                                                                     Treatment     day 1  day 2     day 3                                                                              day 6                                     ______________________________________                                        Saline        0.1    0.6       0.01 -0.01                                       OB Protein -0.9 1.1 0.2 ND                                                    Pegylated Control 0.3 0.4 0.6 -0.2                                            Pegylated OB -0.7 -0.6   -0.9 0.6                                             Protein                                                                     ______________________________________                                    

Data are mean for six ob/ob mice in each group. Mice received a singleIP injection on Day 1 only. Change in body weight is the change in bodyweight on each of the days of the experiment. Note persistent weightloss only in the pegylated OB protein group. ND in the Table indicatesnot determined.

REFERENCES

1. Zhang, Y. et al. Positional cloning of the mouse obese gene and itshuman homologue. Nature 372: 425-432 (1994).

2. Grundy, S. M. & Barnett, J. P. Disease-a-Month 36: 645-696 (1990).

3. Ingalls, A. M. et al. J. Hered. 41: 317-318 (1950).

4. Friedman J. M. et al Genomics 11: 1054-62 (1991).

5. Haley, T. J. and Mc Cormick, W. G. Pharmacological effects producedby intracerebral injection of drugs in conscious mouse. Brit. J.Pharmacol. 12: 12-15 (1957)

6. Sambrook, Fritsch and Maniatis, Molecular Cloning: A LaboratoryManual, Second Edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York).

7. DNA Cloning: A Practical Approach, Vol. I and II (D. N. Glover, ed.1985), MRL Press, Ltd., Oxford, U.K.).

8. Benton and Davis, Science 196: 180 (1977).

9. Grunstein and Hogness, Proc. Nat. Acad. Science 72: 3961 (1975).

10. Pouwels, et al, Cloning Vectors: A Laboratory Manual, (Elsevier, N.J., 1985).

11. Parker and Wiley, Gene 83: 117 (1989)

12. De Sutter, K. et al. Gene 141: 163-170 (1994).

13. Studier et al, J. Mol. Biol. 189: 113 (1986).

14. Lauer, J. Mol. Appl. Genet. 1: 139-147 (1981).

15. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, 1982, p 412).

16. Goeddel et al. Nucleic Acids Research 8, 4057 (1980).

17. Siebenlist et al., Cell 20, 269 (1980).

18. Casadaban, M. J. and Cohen, S. N. Analysis of the gene controlsignals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol.138: 179-207 (1980)),

19. Koshland, D. and Botstein, D.: Secretion of beta-lactamase requiresthe carboxy end of the protein. Cell 20: 749-760 (1980).

20. Laemli, U. K Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227: 680-685 (1970).

21. Matsudaira. P., J. Biol Chem. 262: 10035 (1987).

22. Muurahainen, N. E., et al. Effect of a soup preload on reduction offood intake by cholecystokinin in humans. Am. J. Physiol 260: R672-R680(1991).

23. Drent, M. L., et al. Orlistat (RO 18-0647), a lipase inhibitor, inthe treatment of human obesity: a multiple dose study. Int. J. Obesity19: 221-226 (1995).

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26. Mokhtarian, A, et al Chronic vasacular catherization in the mouse.Physiol. Behav. 54: 895 (1993).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 15                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 702 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CAAGGTGCAA GAAGAAGAAG ATCCCAGGGA GGAAAATGTG CTGGAGACCC CT -            #GTGTCGGT     60                                                                 - - TCCTGTGGCT TTGGTCCTAT CTGTCTTATG TTCAAGCAGT GCCTATCCAG AA -            #AGTCCAGG    120                                                                 - - ATGACACCAA AACCCTCATC AAGACCATTG TCACCAGGAT CAATGACATT TC -            #ACACACGC    180                                                                 - - AGTCGGTATC CGCCAAGCAG AGGGTCACTG GCTTGGACTT CATTCCTGGG CT -            #TCACCCCA    240                                                                 - - TTCTGAGTTT GTCCAAGATG GACCAGACTC TGGCAGTCTA TCAACAGGTC CT -            #CACCAGCC    300                                                                 - - TGCCTTCCCA AAATGTGCTG CAGATAGCCA ATGACCTGGA GAATCTCCGA GA -            #CCTCCTCC    360                                                                 - - ATCTGCTGGC CTTCTCCAAG AGCTGCTCCC TGCCTCAGAC CAGTGGCCTG CA -            #GAAGCCAG    420                                                                 - - AGAGCCTGGA TGGCGTCCTG GAAGCCTCAC TCTACTCCAC AGAGGTGGTG GC -            #TTTGAGCA    480                                                                 - - GGCTGCAGGG CTCTCTGCAG GACATTCTTC AACAGTTGGA TGTTAGCCCT GA -            #ATGCTGAA    540                                                                 - - GTTTCAAAGG CCACCAGGCT CCCAAGAATC ATGTAGAGGG AAGAAACCTT GG -            #CTTCCAGG    600                                                                 - - GGTCTTCAGG AGAAGAGAGC CATGTGCACA CATCCATCAT TCATTTCTCT CC -            #CTCCTGTA    660                                                                 - - GACCACCCAT CCAAAGGCAT GACTCCACAA TGCTTGACTC AA    - #                      - # 702                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 167 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Cys Trp Arg Pro Leu Cys Arg Phe Leu Tr - #p Leu Trp Ser Tyr Leu      1               5   - #                10  - #                15               - - Ser Tyr Val Gln Ala Val Pro Ile Gln Lys Va - #l Gln Asp Asp Thr Lys                  20      - #            25      - #            30                   - - Thr Leu Ile Lys Thr Ile Val Thr Arg Ile As - #n Asp Ile Ser His Thr              35          - #        40          - #        45                       - - Gln Ser Val Ser Ala Lys Gln Arg Val Thr Gl - #y Leu Asp Phe Ile Pro          50              - #    55              - #    60                           - - Gly Leu His Pro Ile Leu Ser Leu Ser Lys Me - #t Asp Gln Thr Leu Ala      65                  - #70                  - #75                  - #80        - - Val Tyr Gln Gln Val Leu Thr Ser Leu Pro Se - #r Gln Asn Val Leu Gln                      85  - #                90  - #                95               - - Ile Ala Asn Asp Leu Glu Asn Leu Arg Asp Le - #u Leu His Leu Leu Ala                  100      - #           105      - #           110                  - - Phe Ser Lys Ser Cys Ser Leu Pro Gln Thr Se - #r Gly Leu Gln Lys Pro              115          - #       120          - #       125                      - - Glu Ser Leu Asp Gly Val Leu Glu Ala Ser Le - #u Tyr Ser Thr Glu Val          130              - #   135              - #   140                          - - Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gl - #n Asp Ile Leu Gln Gln      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Asp Val Ser Pro Glu Cys                                                              165                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Val Pro Ile Gln Lys Val Gln Asp Asp Thr Ly - #s Thr Leu Ile Lys        Thr                                                                             1               5   - #                10  - #                15              - - Ile Val Thr Arg Ile Asn Asp Ile Ser His Th - #r Gln Ser Val Ser Ala                  20      - #            25      - #            30                   - - Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pr - #o Gly Leu His Pro Ile              35          - #        40          - #        45                       - - Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Al - #a Val Tyr Gln Gln Val          50              - #    55              - #    60                           - - Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gl - #n Ile Ala Asn Asp Leu      65                  - #70                  - #75                  - #80        - - Glu Asn Leu Arg Asp Leu Leu His Leu Leu Al - #a Phe Ser Lys Ser Cys                      85  - #                90  - #                95               - - Ser Leu Pro Gln Thr Ser Gly Leu Gln Lys Pr - #o Glu Ser Leu Asp Gly                  100      - #           105      - #           110                  - - Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Va - #l Val Ala Leu Ser Arg              115          - #       120          - #       125                      - - Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gl - #n Leu Asp Val Ser Pro          130              - #   135              - #   140                          - - Glu Cys                                                                  145                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 690 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GTTGCAAGGC CCAAGAAGCC CATCCTGGGA AGGAAAATGC ATTGGGGAAC CC -             #TGTGCGGA     60                                                                 - - TTCTTGTGGC TTTGGCCCTA TCTTTTCTAT GTCCAAGCTG TGCCCATCCA AA -            #AAGTCCAA    120                                                                 - - GATGACACCA AAACCCTCAT CAAGACAATT GTCACCAGGA TCAATGACAT TT -            #CACACACG    180                                                                 - - CAGTCAGTCT CCTCCAAACA GAAAGTCACC GGTTTGGACT TCATTCCTGG GC -            #TCCACCCC    240                                                                 - - ATCCTGACCT TATCCAAGAT GGACCAGACA CTGGCAGTCT ACCAACAGAT CC -            #TCACCAGT    300                                                                 - - ATGCCTTCCA GAAACGTGAT CCAAATATCC AACGACCTGG AGAACCTCCG GG -            #ATCTTCTT    360                                                                 - - CACGTGCTGG CCTTCTCTAA GAGCTGCCAC TTGCCCTGGG CCAGTGGCCT GG -            #AGACCTTG    420                                                                 - - GACAGCCTGG GGGGTGTCCT GGAAGCTTCA GGCTACTCCA CAGAGGTGGT GG -            #CCCTGAGC    480                                                                 - - AGGCTGCAGG GGTCTCTGCA GGACATGCTG TGGCAGCTGG ACCTCAGCCC TG -            #GGTGCTGA    540                                                                 - - GGCCTTGAAG GTCACTCTTC CTGCAAGGAC TACGTTAAGG GAAGGAACTC TG -            #GCTTCCAG    600                                                                 - - GTATCTCCAG GATTGAAGAG CATTGCATGG ACACCCCTTA TCCAGGACTC TG -            #TCAATTTC    660                                                                 - - CCTGACTCCT CTAAGCCACT CTTCCAAAGG         - #                  - #              690                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 167 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Met His Trp Gly Thr Leu Cys Gly Phe Leu Tr - #p Leu Trp Pro Tyr Leu      1               5   - #                10  - #                15               - - Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Va - #l Gln Asp Asp Thr Lys                  20      - #            25      - #            30                   - - Thr Leu Ile Lys Thr Ile Val Thr Arg Ile As - #n Asp Ile Ser His Thr              35          - #        40          - #        45                       - - Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gl - #y Leu Asp Phe Ile Pro          50              - #    55              - #    60                           - - Gly Leu His Pro Ile Leu Thr Leu Ser Lys Me - #t Asp Gln Thr Leu Ala      65                  - #70                  - #75                  - #80        - - Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Se - #r Arg Asn Val Ile Gln                      85  - #                90  - #                95               - - Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Le - #u Leu His Val Leu Ala                  100      - #           105      - #           110                  - - Phe Ser Lys Ser Cys His Leu Pro Trp Ala Se - #r Gly Leu Glu Thr Leu              115          - #       120          - #       125                      - - Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gl - #y Tyr Ser Thr Glu Val          130              - #   135              - #   140                          - - Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gl - #n Asp Met Leu Trp Gln      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Asp Leu Ser Pro Gly Cys                                                              165                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 146 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Val Pro Ile Gln Lys Val Gln Asp Asp Thr Ly - #s Thr Leu Ile Lys        Thr                                                                             1               5   - #                10  - #                15              - - Ile Val Thr Arg Ile Asn Asp Ile Ser His Th - #r Gln Ser Val Ser Ser                  20      - #            25      - #            30                   - - Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pr - #o Gly Leu His Pro Ile              35          - #        40          - #        45                       - - Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Al - #a Val Tyr Gln Gln Ile          50              - #    55              - #    60                           - - Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gl - #n Ile Ser Asn Asp Leu      65                  - #70                  - #75                  - #80        - - Glu Asn Leu Arg Asp Leu Leu His Val Leu Al - #a Phe Ser Lys Ser Cys                      85  - #                90  - #                95               - - His Leu Pro Trp Ala Ser Gly Leu Glu Thr Le - #u Asp Ser Leu Gly Gly                  100      - #           105      - #           110                  - - Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Va - #l Val Ala Leu Ser Arg              115          - #       120          - #       125                      - - Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gl - #n Leu Asp Leu Ser Pro          130              - #   135              - #   140                          - - Gly Cys                                                                  145                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - ATGAAAAAGA CAGCTATCGC GATTGCAGTG GCACTGGCTG GTTTCGCTAC CG -             #TAGCGCAG     60                                                                 - - GCC                  - #                  - #                  - #                 63                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Lys Lys Thr Ala Ile Ala Ile Ala Val Al - #a Leu Ala Gly Phe Ala      1               5   - #                10  - #                15               - - Thr Val Ala Gln Ala                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                               - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GGGTGCTGAG GCCTTGA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                               - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GGGTGCTGAG GCCCTGA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GTGCCTATCC AGAAAGTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Val Pro Ile Glu Lys Val                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - TCCCAAGCTT TCAGCATTCA GGGCTAAC         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GTGCCCATCC AAAAAGTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TCCCAAGCTT TCAGCACCCA GGGCTGAG         - #                  - #                 28                                                                    __________________________________________________________________________

What is claimed is:
 1. A composition comprising one or more conjugates of polyethylene glycol linked to a polypeptide, said polypeptide being a homogeneous purified biologically active human OB protein comprising SEQ ID NO: 6 or a homogeneous purified biologically active murine OB protein comprising SEQ ID NO: 3, characterized in that said polypeptide1) when administered ICV to 16-18 hour fasted mature obese ob/ob mice having a body weight of at least 30 grams at a dose of 20 μg or less,a) reduces food intake during a 5 hour feeding test by 50% compared to vehicle injected control mice (ED50 for reducing food intake); and b) reduces body weight gain during the 24 hours following the ICV injection by at least 50% compared to vehicle injected control mice (ED50 for reducing body weight gain); 2) when administered IP to non-fasted mature ob/ob mice having a body weight of at least 30 grams twice a day at the beginning of daylight and again at the 3 hour point of the dark phase, for one week, in a total daily dose of 10 μg or less,a) reduces 5 and 24 hour food intake by at least 20% compared to vehicle injected control mice (ED20 for reducing food intake); and b) reduces body weight gain during the 24 hours following the first IP injection by at least 20% compared to vehicle injected control mice (ED20 for reducing body weight gain)with the average molecular weight of the polyethylene glycol units in said conjugates within said composition being between 15 kDa to 60 kDa.
 2. The composition of claim 1 wherein said protein is the human OB protein. 